Western Blot Protein Quantification

For protein detection, cells grown for 3 days in a 25 cm² flask were washed with PBS and extracted with 150 µl 1% SDS in TE prior to protein quantification by bicinchoninic acid (BCA) (Pierce). Fifty micrograms of total protein were loaded per gel, and a western blot was performed using M2 anti-Flag mouse monoclonal antibody (Sigma, Cat No. F3165, 1:2000 dilution) or anti-GFP monoclonal antibody (Roche, Cat No. 11814460001, 1:2000 dilution) and detected with anti-mouse IgG conjugated to horseradish peroxidase (Jackson, Cat No. 515035003, 1:10,000 dilution). Blots were developed using high-sensitivity electrochemiluminescence (ECL) reagent (Thermo Fisher Scientific) and visualized using the Fujifilm LAS-3000 developer. Intensities of immunoreactive bands on western blots were quantified using Quantity One (Bio-Rad) software. Protein levels were normalized using a ribosomal protein (RL7) antibody as a loading control^{30 (link)} (1:500 dilution).